The proposed investigation is designed to help elucidate the control or regulation of enzymatic hydroxylation of biological materials using a purified hydroxylase isolated from one of the most prevalent aquatic and soil bacteria, Pseudomonas fluorescens. The enzyme, p- hydroxybenzoate hydroxylase, is representative of an important class of enzymes responsible for the initial reaction in the breakdown of many organic waste materials. The initial objective is elucidation of the mechanism by which the substrate activates the enzyme. The kinetic and thermodynamic properties associated with the substrate activation will be investigated in order to establish the pertinent biophysical characteristics of the enzyme which are necessary for activation and regulatory control of the enzymic activity. While the main objective is to increase our understanding of a class of enzymes involved in an ecologically important task, the degradation of organic waste materials, the bacterial hydroxylase systems may well provide insight into the mechanism of oxygen activation during drug metabolism by the analogous mammalian microsomal system, a system which has proven to be inherently much more difficult to investigate.